IntroductionInactivation of proteins that participate in more than one cellular process leads to a variety of apparently unconnected phenotypes. Understanding the molecular cause for each phenotype might reveal how seemingly independent cellular processes are regulated and coordinated in the cell. Genome-wide gene interaction data based on the simultaneous inactivation of more than one gene greatly facilitate this inherently complex analysis because genes with pleiotropic phenotypes often occupy central positions in the corresponding interaction networks (Costanzo et al., 2010;Tong et al., 2004). By assigning physical connections, protein-protein interaction maps provide the necessary complementary information. Interpretation of these maps is usually not straightforward. Genetic interactions can result from complex functional relationships between the investigated pairs of genes and protein interaction maps are generally projections of contacts that occur at different times and places in the cell. To transform protein interaction data into mechanistically meaningful models, it is necessary to resolve these projections into their different interaction planes. We define an interaction plane or state as the sum of all simultaneously occurring contacts. Ideally, these states should be defined by time-and space-resolved in vivo studies. However, these studies are technically demanding and usually not suited for measuring multiple contacts (Maeder et al., 2007). Using the protein pair Ptc1p-Nbp2p of the yeast Saccharomyces cerevisiae as an example and the split-ubiquitin method (SplitUb) as the experimental tool, we present an alternative approach for defining interaction states. The derived constraint interaction network reduces the number of possible states and thus provides a useful framework for model building and the initiation of more detailed studies.

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These regions are distinguished by an elaborate architecture of proteins and lipids that are specialized to capture and fuse post-Golgi vesicles. Here, we show that the proteins Boi1p and Boi2p are important elements of this area of active exocytosis at the tip of growing yeast cells. Cells lacking Boi1p and Boi2p accumulate secretory vesicles in their buds. The essential PH domains of Boi1p and Boi2p interact with Sec1p, a protein required for SNARE complex formation and vesicle fusion. Sec1p loses its tip localization in cells depleted of Boi1p and Boi2p but overexpression of Sec1p can partially compensate for their loss. The capacity to simultaneously bind phospholipids, Sec1p, multiple subunits of the exocyst, Cdc42p and the module for generating active Cdc42p identify Boi1p and Boi2p as essential mediators between exocytosis and polar growth.

Due to the local enrichment of factors that influence its dynamics, and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 participate in tethering and docking these vesicles to the plasma membrane. Here we show that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip, and shortens their tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2 through their interactions with Bud6 and Bni1 induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane.

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle–specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations.

The Slt2p-Slt2p interaction reported in Fig. 8E was mistakenly not included in Table 1. The authors apologise for this error.